Improved fluorescent Listeria spp. biosensors for analysis of antimicrobials by flow cytometry

Abstract The global increase in antibiotic resistance of pathogenic microorganisms requires the identification and characterization of novel antimicrobials. Bacterial biosensors expressing fluorescent proteins such as pHluorin variants are suitable for high‐throughput screenings. Here, we present Listeria spp. pH‐sensitive biosensors with improved fluorescence for single‐cell analysis of antimicrobials by flow cytometry.

The increasing global challenges with (multi)drug-resistant bacteria highlight the demand for novel antimicrobial compounds to treat lifethreatening infections (World Health Organization, 2014). Despite this growing need for novel anti-infective agents, the number of new antibiotics on the market is steadily decreasing (Theuretzbacher et al., 2020;Towse et al., 2017). A major bottleneck in the development of new antimicrobial drugs is the lack of rapid, costeffective, and reliable screening tools for lead compound identification (Miethke et al., 2021). Recently, our group has developed live a biosensor of the food-borne pathogen Listeria monocytogenes for the detection of antimicrobial compounds that kill target bacteria by pore formation and disruption of membrane integrity (Crauwels et al., 2018). The biosensor is based on monitoring intracellular pH by expression of the green fluorescent protein-derivative pHluorin, which is characterized by two distinct excitation peaks that change in relative fluorescence intensities in response to pH (Miesenböck et al., 1998). These biosensors were successfully used to determine the susceptibility of bacteria to the lantibiotic nisin, measure antimicrobial activity in supernatants for natural and recombinant producers of antimicrobial peptides, and screen a library of bacteria isolated from raw milk for producers of antimicrobials (Desiderato et al., 2021;Goldbeck et al., 2021;Weixler et al., 2022).
Similar to the previously published biosensor strain L. monocytogenes EGDe/pNZ-P help -pHluorin (Lm pHin), a new vector was constructed, in which the pHluorin gene was replaced with a gene for pHluorin2, a pHluorin derivative with enhanced fluorescence (Mahon, 2011). The backbone of pNZ44 (McGrath et al., 2001) was linearized by restriction with BglII and PstI (FastDigest enzymes, Thermo Fisher Scientific) to remove the p44 promoter. The strong, constitutive P help promoter was amplified from pPL2luxP help (Riedel et al., 2007) using primers P help _fw (TTTTTATATTACAGCTCCAAT CATTATGCTTTGGCAGTTTATTC) and P help _rv (CTTTACTCATG GGTTTCACTCTCCTTCTAC) using Q5 polymerase (New England Biolabs) and a standard PCR protocol with 61.9°C annealing temperature and 15 s elongation time. The gene encoding pHluorin2 was obtained as a synthetic DNA fragment codon-optimized for L. monocytogenes by a commercial service provider (Eurofins Genomics) and amplified using primers pHin2LM_fw (GTAGAAGGAGAGTG AAACCCATGAGTAAAGGTGAAGAATTATTTAC) and pHin2LM_rv (AGTGGTACCGCATGCCTGCACTATTTATATAATTCATCCATACCAT GTG) (Q5 polymerase, 58.6°C annealing, 45 s elongation). Vector backbone and PCR products were assembled in a single isothermal reaction as described by Gibson et al. (2009) www.MicrobiologyOpen.com sequencing (Microsynth Seqlab). Correct plasmids were used to transform L. monocytogenes EGDe and Listeria innocua LMG 2785 by electroporation using a previously described protocol (Monk et al., 2008) and positive clones were selected on brain heart infusion (BHI) agar containing 10 µg/ml chloramphenicol. Following successful transformation, biosensor strains carrying plasmids pNZ-P help -pHluorin or pNZ-pHin2 Lm were initially checked for fluorescence by imaging in an iBright TM FL1000 Imaging System (Thermo Fisher Scientific) with fluorescence detection mode at 488 nm ( Figure 1b).
Both new strains containing pNZ-pHin2 Lm (Li pHin2, Lm pHin2) showed brighter fluorescence on agar plates than the previously published strain Lm pHin, whereas the empty vector control strain L.
To further analyze the fluorescence properties of the biosensors, bacteria were grown in BHI overnight (i.e., approx. 16 h), washed once in phosphate-buffered saline (PBS), and adjusted to an OD 600 of 3 in filter-sterilized (pore size 0.2 μm) Listeria minimal buffer (LMB) (Crauwels et al., 2018) adjusted to different pH (5.5-8.5). Aliquots of 100 μl were distributed into single wells of a black microtiter plate and mixed with 100 μl of LMB containing the cationic detergent cetyltrimethylammonium bromide (final concentration 0.002% w/v) for membrane disruption. After incubation for 30 min at room temperature, fluorescence excitation spectra (350-490 nm) were recorded at an emission wavelength of 520 nm using a Tecan Infinite ® M200 multimode plate reader (Tecan).
monocytogenes EGDe/pNZ-pHin2 Lm (Lm pHin2) displayed the typical excitation spectrum of pHluorin proteins with excitation peaks at 400 and 475-480 nm ( Figure 2). All three strains also showed the characteristic ratiometric, pH-dependent shift in fluorescence intensities across the excitation spectrum. However, fluorescence intensities were up to 6.7-and 9-fold higher for Li pHin2 Lm and Lm pHin2 Lm compared to Lm pHin depending on excitation wavelength and pH ( Figure 2). This is in line with data showing about 8-fold higher fluorescence for pHluorin2 over pHluorin when expressed in eukaryotic cells (Mahon, 2011).
To further demonstrate that the new biosensors behave similarly to the previously published strain, dose-response experiments were performed with nisin A and pediocin PA-1 (both purchased from Sigma-Aldrich), two antimicrobial peptides that kill target bacteria by disrupting membrane integrity (Brötz et al., 1998;Chikindas et al., 1993). In particular, 2-fold dilutions of the peptides were prepared in microtiter plates, starting with concentrations of 10 µg/ml nisin and 1.25 µg/ml pediocin. The sensor strains were prepared as described above and 100 µl aliquots were added to Bacteria in these two gates either represent untreated, intact, or nisin-treated, membrane-disrupted bacteria, respectively. This demonstrates that flow cytometry can be used to assess intracellular pH and in consequence membrane integrity of pHluorin-expressing sensor bacteria on a single-cell level.
In conclusion, we provide two new biosensors of the genus Listeria that allow the analysis of membrane damage using the ratiometric pH-dependent fluorescent protein pHluorin2 (Mahon, 2011). Both strains show up to 9-fold higher fluorescence compared to previously published strain L. monocytogenes EGDe/ pNZ-P help -pHluorin (Crauwels et al., 2018

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
The data generated or analyzed during this study are included in the published article. The sequence of pNZ-pHin2 Lm is available in GenBank, accession number ON668434: https://www.ncbi.nlm.nih. gov/nuccore/ON668434.

ETHICS STATEMENT
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